Tanshinones and phenolic acids are two important metabolites synthesized by the traditional Chinese medicinal plant Salvia miltiorrhiza . There is increasing market demand for these compounds. Here, we isolated and functionally characterized SmERF1L1 , a novel JA (Jasmonic acid)-responsive gene encoding AP2/ERF transcription factor, from Salvia miltiorrhiza . SmERF1L1 was responsive to methyl jasmonate (MJ), yeast extraction (YE), salicylic acid (SA) and ethylene treatments. Subcellular localization assay indicated that SmERF1L1 located in the nucleus. Overexpression of SmERF1L1 significantly increased tanshinones production in transgenic S. miltiorrhiza hairy roots by comprehensively upregulating tanshinone biosynthetic pathway genes, especially SmDXR . Yeast one-hybrid (Y1H) and electrophoretic mobility shift assay (EMSA) showed that SmERF1L1 binds to the GCC-box of SmDXR promoter while dual luciferase (Dual-LUC) assay showed that SmERF1L1 positively regulated the expression of SmDXR . Our study suggested that the SmERF1L1 may be a good potential target for further metabolic engineering of bioactive component biosynthesis in S. miltiorrhiza .
Deng Changping,Shi Min,Fu Rong,Zhang Yi,Wang Qiang,Zhou Yang,Wang Yao,Ma Xingyuan,Kai Guoyin
Phenolic acids and tanshinones are major bioactive ingredients in Salvia miltiorrhiza, which possess pharmacological activities with great market demand. However, transcriptional regulation of phenolic acid and tanshinone biosynthesis remains poorly understood. Here, a basic leucine zipper transcription factor (TF) named SmbZIP1 was screened from the abscisic acid (ABA)-induced transcriptome library. Overexpression of SmbZIP1 positively promoted phenolic acid biosynthesis by enhancing expression of biosynthetic genes such as cinnamate-4-hydroxylase (C4H1). Furthermore, biochemical experiments revealed that SmbZIP1 bound the G-Box-like1 element in the promoter of the C4H1 gene. Meanwhile, SmbZIP1 inhibited accumulation of tanshinones mainly by suppressing the expression of biosynthetic genes including geranylgeranyl diphosphate synthase (GGPPS) which was confirmed as a target gene by in vitro and in vivo experiments. In contrast, the phenolic acid content was reduced and tanshinone was enhanced in CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9]-mediated knockout lines. In addition, the previously reported positive regulator of tanshinone biosynthesis, SmERF1L1, was found to be inhibited in SmbZIP1 overexpression lines indicated by RNA sequencing, and was proven to be the target of SmbZIP1. In summary, this work uncovers a novel regulator and deepens our understanding of the transcriptional and regulatory mechanisms of phenolic acid and tanshinone biosynthesis, and also sheds new light on metabolic engineering in S. miltiorrhiza.
危险品化学品经营许可证（带存储）